Hemodynamic shear stress stimulates number of intracellular events that both regulate vessel structure and also influence development of vascular pathologies. The precise molecular mechanisms by which endothelial cells transduce this mechanical stimulus into intracellular biochemical response have not been established yet. The central hypothesis is that the plasma membrane of endothelial cell acts as a mechanosensitive element; i.e. changes in physical properties of the membrane under mechanical stress can regulate activity of membrane proteins coupled to intracellular signaling pathways. To test this hypothesis, we will use an integrative approach that combines time-resolved fluorescence microscopy, biochemistry, cell biology, and membrane micromechanics. Our preliminary experiments show for the first time that (1) when exposed to mechanical forces, membrane lateral fluidity and hydration levels change and (2) that increases in membrane tension lead to activation of bradykinin G protein coupled receptor (GPCR). The proposed research addresses the following questions: (1) which physical properties of the lipid bilayer change in response to mechanical perturbation, (2) which of these changes has a clear link to function of membrane-associated proteins such as GPCRs, G-proteins and endothelial nitric oxide synthase (eNOS), and can mediate mechanochemical signal transduction, and (3) what are the specific mechanisms leading to mechanically induced activation of GPCR receptors, eNOS and G-proteins by shear stress. We will use state-of-the-art picosecond time-resolved fluorescence, single molecule and fluorescence correlation spectroscopy techniques to investigate in detail what happens to the physical properties of the lipid bilayer membrane at the molecular level under mechanical stress and how these changes are coupled to mechanochemical signal transduction via direct activation of the membrane associated proteins such as GPCR's and modulation of signal amplification cascades through G- proteins. Specifically we propose that mechanically-induced changes in certain membrane properties such as thickness, lateral fluidity, polarity, membrane free volume and/or trans-membrane lateral force profile are able to initiate and regulate conformational changes responsible for experimentally observed response of GPCR and G protein signal transduction pathways and eNOS activation. If successful it will provide the mechanistic basis on how endothelial cells sense flow in both normal physiology and in vascular disease.